Preservation and storage of biologic materials

ABSTRACT

Method for preserving and storing biologic material including viable beings, living tissue and organs, foodstuffs and the like. The biologic material is subjected to ultrahyperbaric pressure of about 2.0 X 103 atmospheres in the presence of inert gas. The temperature is reduced to about -20* C., whereupon the material is subjected to a further pressure increase to below about 3.4 X 103 atmospheres to effect a phase change of water from the liquid phase to the solid phase. The temperature and pressure of the material is then reduced to about atmospheric pressure at the temperature necessary to maintain the formed solid phase of water. Under such conditions the materials may be stored for extended periods of time without significant deterioration. The process of preservation is reversed to restore the material to ambient conditions.

[54] PRESERVATION AND STORAGE OF BIOLOGIC MATERIALS Paul E. Segall, 15Cambridge Road, East Rockaway, NY. 11518 i221 Filed: Jan.20, 1970 21Appl.No.: 4,430

[ 72] Inventor:

[52] US. Cl ..62/64, 99/189, 128/1,

195/ 1.8 [51] Int. Cl ..F25d 17/02 [58] Field of Search ..62/62, 4, 66,56,75; 128/1; 195/17, 1.8; 99/197, 8,189,192

[56] References Cited UNITED STATES PATENTS 2,875,588 3/1959 Berger..62/64 X 2,662,520 12/1953 McMahon 3,092,974 6/1963 Haumann eta]..62/62 [451 July 18, 1972 Primary Examiner-Meyer Perlin AssistantExaminerRonald C. Capossela AttorneyMarn and .langarathis ABSTRACTMethod for preserving and storing biologic material including viablebeings, living tissue and organs, foodstuffs and the like. The biologicmaterial is subjected to ultrahyperbaric pressure of about 2.0 X 10atmospheres in the presence of inert gas. The temperature is reduced toabout -20 C., whereupon the material is subjected to a further pressureincrease to below about 3.4 X 10 atmospheres to effect a phase change ofwater from the liquid phase to the solid phase. The temperature andpressure of the material is then reduced to about atmospheric pressureat the temperature necessary to maintain the formed solid phase ofwater. Under such conditions the materials may be stored for extendedperiods of time without significant deterioration. The process ofpreservation is reversed to restore the material to ambient conditions.

15 Claims, No Drawings PRESERVATION AND STORAGE OF BIOLOGIC MATERIALSThis invention relates to the preservation of biologic material, andmore particularly to the preservation and storage of biologic materialsfor extended periods of time without significant deleterious effects.

Biologic materials are composed to a considerable extend of cellularcomponents containing aqueous fluid. Such materials are difficult topreserve and store for any substantial length of time, as is apparentfrom the spoilage of food; deterioration of shelf life of medicines andbiochemicals; and the aging of the human body. The deterioration andother detrimental reactions which occur quite rapidly in these materialsat ordinary room temperatures can be greatly slowed as the materials aresubjected to lower and lower temperature, and theoretically arrested atabsolute zero. Even with biochemical materials stored at C., it isnecessary to use such materials as soon as possible before deteriorationbecomes significant. It has been suggested to preserve and store suchmaterials at below normal freezing temperatures under hydrostaticallyinduced high pressures.

For several decades meat, fish, vegetables and fruit have I beenavailable at frozen food counters, but have met with varying degrees ofpublic satisfaction. Early attempts resulted in considerable damage tothe product during freezing with a concomitant reduction in flavor,which is overcome, even today, by the addition of artificial flavoring.While advances have been made in the coffee industry, i.e., freeze driedcoffee, the freezing of nutritional foods still suffer from damage byfreezing with loss of flavor and consistency necessitating the additionof chemical preservatives and flavoring.

Water and aqueous solutions upon being subjected to loweringtemperatures expand upon freezing. Consequentially, materials fonned ofor containing cells when subjected to lowering temperatures, atatmospheric pressure, are destroyed by rupturing of the cells which isthe reason that known freezing process for the preservation and storageof biological materials leave room for improvement.

It is an object of my invention to provide an improved method forpreserving a biologic material in substantially a solid phase system.

Another object of my invention is to provide a method for preserving andstoring a biologic material in a solid phase system at atmosphericpressure for extended periods of time.

Still another object of my invention is to revive a biologic materialstored in a solid phase system at atmospheric pressure to substantiallythe condition the biologic material existed prior to preservation andstorage without significant deterioration.

A further object of my invention is to provide a process for preservingand storage of biologic material which requires no chemicalpreservatives.

Still another object of my invention is to provide a process for thepreservation and storage of foodstuffs which may be restored to acondition not requiring artificial flavoring.

These and other objects of the invention shall become readily apparentfrom the following description.

The biologic materials which may be treated in accordance with thisinvention include various living biological materials, such as tissues,whole organisms, and organs. Organs include livers, hearts, lungs andother tissues suitable for transplanting. Additionally, foodstuffs, suchas fruits, vegetables, meats, fishes and the like may be treated inaccordance with the invention. Additionally, non-living biologicalmaterials, such as vaccines, sera, enzymes and hormones may beprocessed. Thus, it will no longer be necessary to rely entirely uponseasonal supply of fresh fruits and vegetables; year-round supply ofsuch fruits and vegetables can be made available. Seafoods, such aswhole lobsters and crabs, may be preserved and stored when caught withflavor intact, since substantially no cellular damage is effected by themethod of this invention. The biologic material treated may be storedindefinitely without significant histological, biochemical orphysiological deterioration.

In accordance with one embodiment of the invention, the biologicmaterial to be processed is placed in a chamber constructed to withstandultrahyperbaric pressures, i.e., pressures exceeding about 2.0 X 10atmospheres and cryogenic temperatures, i.e., about 200 C., and which isprovided with suitable pressure and temperature control means.Temperature control within the chamber may be effected by placing thechamber within a vessel through which a heat transfer medium is passed,or by providing indirect heat transfer coils about the chamber walls,within the chamber walls, or inside the chamber, or any combination ofsuch means. pressurization of the chamber is achieved by the use of amulti-stage gas compressor or by liquid under pressure.

The chamber is purged with an inert gas and then pressurized with suchinert gas at a rate to allow all components of the material to reachequilibrium. Any inert gas or liquid (fluid) may be utilized which doesnot react with and thus alter the material being processed. Helium isparticularly useful being monoatomic with a small diameter and mass, andis readily permeable to the various membranes comprising the cells,tissue, etc., of the biologic material. It is contemplated that othernoble gases may be used, such as neon and argon. It is understood thatby using gases heavier than helium, that time factors will be differentsince such heavier gases will permeate such membranes at lower ratesthereby more slowly reaching an equilibrium condition.

The pressure within the chamber is then raised to about 2.0 X 10atmospheres by the compressor as the temperature is lowered to about 20C. The pressure may be first raised with the temperature beingsubsequently lowered, or both temperature and pressure may be loweredand raised, respectively, provided that there is no formation of a solidphase of any aqueous systems.

Upon reaching these conditions the pressure is preferably thereafterslowly raised to a pressure less than about 3.4 X 10" atmosphereswhereby the aqueous systems of the biologic material undergoes a rapid,gradient free transition into a solid state system. Under theseconditions, slight contraction occurs of the solid phase of any aqueoussystem, with substantially no damage to the cellular components of thebiologic material. Altemately, the temperature may be lowered at 2.0 X10 atmospheres to cause the aqueous systems to undergo the phase changeto the solid state system. Effecting the phase change by loweringtemperature would involve temperature gradients, which for thepreservation of foodstufls and non-living biologic materials would beacceptable. For living biological materials, however, it is preferred toeffect the phase change by increasing the pressure since a gradient freechange is effected substantially instantaneously throughout the entirematerial.

In accordance with another embodiment of the invention, the temperaturewithin the chamber is further reduced with the pressure thereafter beingreduced to atmospheric. The temperature to which the material is loweredis that temperature at which no phase change of the solid state systemwould occur when the pressure is lowered to atmospheric pressure. Liquidnitrogen (i.e., 196 C.) being readily available, is a particularlysuitable refrigerant to lower the temperature of the biologic material.Additionally, after reduction of pressure to atmospheric, the materialmay be exposed to liquid nitrogen without deleterious effects sinceliquid nitrogen (at atmospheric pressure) is substantially inert. Thenow frozen biologic material may then be transferred to another vesselat atmospheric pressure and maintained at low temperatures by liquidnitrogen (-1 96 C. wherein the material may be stored for almostindefinite periods of time at relatively low costs.

When desired, the biologic material may be returned to ambientconditions by reversing the procedure. Accordingly, the frozen biologicmaterial is placed in the ultrahyperbaric chamber, which after closure,is purged with the inert gas used to effect preservation, such ashelium. The chamber is then pressurized with the inert gas to a pressureof about 3.0 X 10 atmospheres and the temperature raised to about 20 C.,

while maintaining the solid phase system. Upon reaching theseconditions, the pressure is slowly reduced to about 2.0 X atmosphereswhereby a gradient free and substantially instantaneous change iseffected from a solid phase system to a liquid phase system. Thereafterthe pressure is further reduced to atmospheric pressure with aconcomitant increase in temperature controlled to prevent any change ofany aqueous system to a solid phase.

when preserving living whole organisms or organs it is desirable toinitially reduce the temperature of such whole organisms or organs to atemperature of minimal activity. Such a temperature is about 4 C., whichis also the temperature of maximum density of water in the aqueoussystems. Such initial temperature will vary for the diverse organs orwhole organisms being treated.

The method of this invention has four major advantages over processesfor the freezing of biologic materials at atmospheric pressures. First,there is substantially no destructive expansion during the liquidstate-solid state transition (crystallization). Secondly, since thepreferred method depends upon pressure for crystallization, it isthermally gradient free, so that large masses may be frozenhomogeneously. Thirdly, crystallization, being very rapid andhomogeneous, allows suspended and dissolved particles to be frozen intheir natural state, rather than in s salted out condition. Fourthly,helium, under 30,000 psi, acts as an efficient heat conductor, and thuswould allow for rapid and even thermal changes.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, it is to beunderstood that the invention may be practiced otherwise than asparticularly described.

lclaim:

1. A method for preserving biologic material having aqueous systemswhich comprises:

a. placing said biologic material into a hyperbaric chamber;

b. introducing an inert fluid into said chamber in direct contact withsaid biologic material and pressurizing said chamber with said fluid toa pressure of about 2.0 X 10 atmospheres; and

0. further reducing the temperature in said chamber and furtherincreasing the pressure in said chamber to change the fluid phase ofsaid aqueous systems to a solid phase.

2. The method defined in claim 1 wherein the temperature reduction ofstep (c) is to a temperature of -20 C. and thereafter maintainedconstant as the pressure is increased.

3. The method defined in claim 2 wherein the pressure is increased to apressure below about 3.4 X 10 atmospheres.

4. The method defined in claim 1 wherein the chamber is purged with aninert gas prior to pressurizing said chamber with said inert gas.

5. The method as defined in claim 1 wherein the temperature of thebiologic material to be treated is reduced to a temperature of minimalactivity, about 4 C., at atmospheric pressure prior to introduction intosaid chamber.

6. The method as defined in claim 1 wherein helium is the 7. A methodfor preserving and storing biologic material having aqueous systemswhich comprises:

a. placing said biologic material into a hyperbaric chamber;

b. introducing an inert fluid into said chamber in direct contact withsaid biologic material and pressurizing said chamber to a pressure ofabout 2.0 X 10 atmospheres;

c. reducing the temperatures in said chamber;

d. increasing the pressure in said chamber to change the fluid phase ofsaid aqueous systems to a solid phase;

e. further reducing the temperature and pressure in said chamber whilemaintaining the solid phase of said aqueous systems, said pressure beingreduced to about atmospheric; and

f. storing said material at about atmospheric pressure at thetemperature necessary to maintain said solid phase.

8. The method defined in claim 7 wherein the temperature of the biologicmaterial is reducedto a temperature of minimal activity, about 4 C.,prior to introduction into said chamber.

9. The method as defined in claim 7 wherein the temperature reduction ofstep (c) is to a temperature of -20 C. and maintained constant prior tosaid pressure increase.

10. The method as defined in claim 9 wherein the pressure is increasedto a pressure less than about 3.4 X 10 atmospheres.

11. The method as defined in claim 7 wherein helium is the inert fluid.

12. The method as defined in claim 7 wherein the temperature reductionof step (e) is to a temperature of 1 96 C.

13. The method defined in claim 11 wherein the temperature reduction ofstep (e) is effected prior to pressure reduction.

14. The method as defined in claim 7 wherein the material is stored at atemperature at which nitrogen is a liquid.

15. A method for restoring the viability of a biologic material storedin accordance with claim 7 which comprises a. subjecting said materialto a pressure of between about 2.2 to 3.4 X 10 atmospheres;

b. thereupon raising the temperature of said material to a temperatureof at least about 20 C.;

c. reducing the pressure on said material to about 2.0 X 10 atmosphereswhereupon the solid phase changes to a liquid phase; and

d. returning the material to ambient conditions while avoiding formationof another solid phase of said aqueous systems.

2. The method defined in claim 1 wherein the temperature reduction ofstep (c) is to a temperature of -20* C. and thereafter maintainedconstant as the pressure is increased.
 3. The method defined in claim 2wherein the pressure is increased to a pressure below about 3.4 X 103atmospheres.
 4. The method defined in claim 1 wherein the chamber ispurged with an inert gas prior to pressurizing said chamber with saidinert gas.
 5. The method as defined in claim 1 wherein the temperatureof the biologic material to be treated is reduced to a temperature ofminimal activity, about 4* C., at atmospheric pressure prior tointroduction into said chamber.
 6. The method as defined in claim 1wherein helium is the inert fluid.
 7. A method for preserving andstoring biologic material having aqueous systems which comprises: a.placing said biologic material into a hyperbaric chamber; b. introducingan inert fluid into said chamber in direct contact with said biologicmaterial and pressurizing said chamber to a pressure of about 2.0 X 103atmospheres; c. reducing the temperatures in said chamber; d. increasingthe pressure in said chamber to change the fluid phase of said aqueoussystems to a solid phase; e. further reducing the temperature andpressure in said chamber while maintaining the solid phase of saidaqueous systems, said pressure being reduced to about atmospheric; andf. storing said material at about atmospheric pressure at thetemperature necessary to maintain said solid phase.
 8. The methoddefined in claim 7 wherein the temperature of the biologic material isreduced to a temperature of minimal activity, about 4* C., prior tointroduction into said chamber.
 9. The method as defined in claim 7wherein the temperature reduction of step (c) is to a temperature of-20* C. and maintained constant prior to said pressure increase.
 10. Themethod as defined in claim 9 wherein the pressure is increased to apressure less than about 3.4 X 103 atmospheres.
 11. The method asdefined in claim 7 wherein helium is the inert fluid.
 12. The method asdefined in claim 7 wherein the temperature reduction of step (e) is to atemperature of -196* C.
 13. The method defined in claim 11 wherein thetemperature reduction of step (e) is effected prior to pressurereduction.
 14. The method as defined in claim 7 wherein the material isstored at a temperature at which nitrogen is a liquid.
 15. A method forrestoring the viability of a biologic material stored in accordance withclaim 7 which comprises a. subjecting said material to a pressure ofbetween about 2.2 to 3.4 X 103 atmospheres; b. thereupon raising thetemperature of said material to a temperature of at least about -20* C.;c. reducing the pressure on said material to about 2.0 X 103 atmosphereswhereupon the solid phase changes to a liquid phase; and d. returningthe material to ambient conditions while avoiding formation of anothersolid phase of said aqueous systems.